Glial cells monitor and respond to neural activity by conditioning the extacellular milieu, signaling within glial cell networks as well as by sending signals back to neurons. Unlike neurons, which use electrical signals to communicate, glial cells possess a form of Ca2+ based excitability, where they generate and propagate intracellular Ca2+ signals as waves over long distances in response to monitored synaptic activity. We aim to understand the principles and mechanisms that govern intracellular calcium signals in glial cells and neurons. One objective is to understand processes that support temporal and spatial characteristics of Ca2+ signals within cells and between cells. A second objective is to understand the precise nature of glial cell signals in response to neuronal activity and the consequence of such signals to CNS function. In previous work, we found that wave propagation was saltatory due to the underlying regenerative Ca2+ release process. Regenerative Ca2+ release occurs at wave amplification sites, which are specialized Ca2+ release sites found 5 to 7 micrometers apart along glial cell processes. We have recently characterized the kinetics of Ca2+ release, at presumed wave amplification sites (microdomains of specialized Ca2+ release aites) by recording and analyzing Ca2+ sparks and Ca2+ puffs in glial cell processes. A number of important findings resulted from these series of experiments: 1. These sites are characterized by high-density patches of endoplasmic reticulum (ER) proteins such as the inositol 1,4,5-trisphosphate receptors (IP3Rs), sarco-endoplasmic reticulum calcium pumps, calreticulin and at least one mitochondrion in close association. 2. Ca2+ release at these sites occurs through IP3R ion channels. 3. This release is modulated by the ryanodine receptor channels, which are also found in close proximity to IP3Rs. 4. Mitochondrial transport processes also modulate this Ca2+ release process. Based on these findings we hypothesize that the wave amplification sites are indeed specialized microdomains of Ca2+ release. At these sites a number of specialized proteins involved in aspects of Ca2+ signaling closely interact to form a complex protein machine. Some of these protein components are present in different membrane systems. These include the plasma membrane, the endoplasmic reticulum membrane and mitochondrial membrane. In addition, we hypothesize that such sites are organized in cellular processes at strategic locations in order to serve the signaling needs of the cell. This anchoring occurs by specific interaction of signaling proteins with molecular scaffolds and adapter proteins, which anchor the entire assembly to cytoskeletal components. Current research is focused on a complete molecular characterization of this putative signaling microdomain using modern proteomic technologies. We are in the process of isolating Ca2+ signaling microdomains from oligodendrocyte progenitor cells as well as astrocytes from mouse brains. Oligomeric protein complexes will be separated using immunoaffinity strategies and will be analyzed using proteomic methods. Two approaches will be used. In one, mass spectra of proteolytic digests of proteins spots excised from electrophoresis gels will be analyzed by direct mass spectrometry. MALDI-TOF will be used for initial peptide mass finger printing. Masses of peptides derived from an in-gel proteolytic digestion will be measured and searched against a computer-generated list formed from the simulated digestion of a protein database using the same enzyme. In the second approach, proteins whose full-length sequences are not yet available will be analyzed using a nano-electrospray technology. Proteolytic peptide pools of unknown proteins will be separated by liquid chromatography followed by tandem electrospray mass spectrometry (LC-MS/MS). The electrospray technique generates peptide sequence tags, which enable searching in EST data bases of partially sequenced proteins. A comprehensive description of the component protein machines that comprise signaling microdomains in glial cells is expected to result from this investigative effort. We then plan to investigate the importance of protein-protein interactions in signaling. We will begin by characterizing the cellular localization of selected protein components using immunocytochemistry. Furthermore, we will use protein expression mapping to compare different functional states of cells in order to study global changes in expression of specific components of signaling microdomains. Finally, we will use strategies to inhibit protein-protein interactions or suppressing expression by gene knock out, antisense RNA or dominant negative approaches to query functional consequences. Our long-term goal is to describe in detail the nature of communication between neuronal networks and glial cell networks. We are developing experimental models to investigate the physiological consequences of glial cell signals in response to neuronal activity. Impaired glial cell signaling has been implicated in a number of pathological states in the CNS such as excitotoxicity, brain edema and certain degenerative diseases. It is hoped that a detailed understanding of the glial cell signaling modes will be useful in understanding the pathophysiology of such conditions.